Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Biochimica et Biophysica Acta 1576 (2002) 119 – 126 www.bba-direct.com Purification, characterization, and genetic analysis of a leucine aminopeptida...

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Biochimica et Biophysica Acta 1576 (2002) 119 – 126 www.bba-direct.com

Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae Hung-Chien Roger Chien a, Long-Liu Lin b, Shiou-Huei Chao c, Chun-Chang Chen c, Wen-Ching Wang d, Chin-Ying Shaw e, Ying-Chie Tsai e, Hui-Yu Hu b, Wen-Hwei Hsu c,* a Department of Microbiology, Chung Shan Medical University, Taichung 402, Taiwan Department of Food Science and Nutrition, Hung Kuang Institute of Technology, Taichung 433, Taiwan c Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan d Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan e Institute of Biochemistry, National Yang Ming University, Taipei 112, Taiwan

b

Received 7 November 2001; received in revised form 27 February 2002; accepted 21 March 2002

Abstract Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 jC for 30 min. The enzyme preferentially hydrolyzed leucine pnitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn2 + and Co2 + . The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11 – 33.1% identity with those of LAPs from 18 organisms. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Leucine aminopeptidase; lap gene; Aspergillus sojae; Glycosylation

1. Introduction Leucine aminopeptidase (LAP) is a prototypic dizinc exopeptidase that selectively removes N-terminal amino acid residues from polypeptides and proteins [1 – 3]. In mammals, LAP is present as a hexameric oligomer [4]. Although the real role for this enzyme in mammalian cells is still to be elucidated, changes in LAP activity have been correlated with lens aging [5] and hepatic diseases [6]. Moreover, it has been proposed that the mammalian enzymes are involved in the turnover of normal and/or damaged proteins and peptides based on the constitutional expression of mRNAs and LAP activity seen in all organs examined [7]. Studies of LAPs from Arabidopsis and *

Corresponding author. Tel.: +886-4-2285-1885; fax: +886-4-22874879. E-mail address: [email protected] (W.-H. Hsu).

tomato also reveal constitutive expression, suggesting a similar catabolic role in plants [8,9]. Additional roles for plant LAPs have been correlated with mechanical wounding, insect feeding, and pathogen attack [9,10]. LAPs are also found in a wide variety of microbial species including bacteria and fungi [11]. Despite that most microbial LAPs are intracellular enzymes, extracellular enzymes are found in Aeromonas proteolytica [12], Pseudomonas aeruginosa [13], Streptomyces griseus [14], and a filamentous fungus Aspergillus oryzae [15 – 18]. Crystal structures of A. proteolytica and S. griseus LAPs show that LAP is a monomeric metalloenzyme with a globular a/h domain [19,20], and two zinc ions situated closely together near the catalytic center [19]. These two microbial enzymes differ from bovine lens LAP in both overall structure and in coordination of the two zinc ions [2]. The intensity of the bitterness for a peptide is proportional to the number of hydrophobic amino acids [21].

0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 2 ) 0 0 3 0 7 - X

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Based on the fact that LAP can remove single or pairs of hydrophobic amino acids from the N-terminus of a polypeptide chain, it is considered to be useful for debittering protein hydrolysates with N-terminal hydrophobic amino acids, which are commonly used as clinical nutrition supplements [22,23]. Besides, LAP can improve flavor development [24] and convert L-homophenylalanyl amide into Lhomophenylalanine, the versatile intermediate for a class of angiotension 1-converting enzyme inhibitors, such as Enalapril, and Benzapril [25]. Currently, commercial LAPs used to reduce or eliminate bitterness are from Lactobacillus lactis, Rhizopus oryzae, Aspergillus sojae, and A. oryzae. Among these species, filamentous fungi of A. oryzae and A. sojae have a long history of safe use as koji mold in the manufacture of soy sauce. Although four A. oryzae LAPs have been purified and characterized (26.5, 37.5, 56, and 61 kDa, respectively) [15 –18], gene structures of these proteins are not known. In this investigation, we describe the purification and characterization of an A. sojae LAP. Furthermore, we report the lap gene from its genomic DNA and cDNA by cloning and sequencing. To our knowledge, this is the first genetic analysis on the LAP from the genus Aspergillus.

with a NaCl gradient of 0 to 1 M in the 20 mM Tris/HCl buffer (pH 7.5). The active fractions were pooled and dialyzed against 20 mM sodium phosphate buffer (pH 6.5). The pooled sample was applied to a Mono Q 5/5 High Performance column (Amersham Pharmacia), and then eluted with a linear gradient of 0 –0.15 M NaCl in 20 mM sodium phosphate (pH 6.5) followed by two-step gradients at 0.15– 0.3 and 0.3– 1 M NaCl in the same buffer. Under this purification procedure, the LAP was eluted in a sharp and symmetrical active peak. For amino acid sequence determination, the purified A. sojae LAP was cleaved with lysylendopeptidase at 42 jC for 18 h. Peptides were injected onto an HPLC narrowbore column (Vydac C18) and eluted with an acetonitrile gradient of 0% to 80% in 0.07% trifluoroacetic acid. Seven internal peptides were selected and electroblotted onto a PreSorb Immobilon polyvinylidene difluoride membrane (PEApplied Biosystems, Foster City, CA), and the N-terminal amino acids of the endopeptidase-cleaved fragments were determined by Edman degradation method [26] with an Applied Biosystems 475A gas phase (PE-Applied Biosystems). 2.3. Enzyme assay

2. Materials and methods 2.1. Microorganisms, plasmids, and culture conditions A. sojae ATCC 42249, an industrial strain for soy sauce production, was used as the donor of mRNA and DNA. Escherichia coli XL1 Blue MRFV (recA1 endA1 gyrA96 thi hsdR17 (rk mk + ) supE44 relA1 lac (FVproAB + lacI qZD M15DTn10)); (Stratagene, La Jolla, CA) was used for construction of the A. sojae genomic library and for DNA manipulations. The plasmid pBluescript KS (+) (Stratagene) was the vector used for DNA fragment cloning. A. sojae was grown at 30 jC in a complete medium (CM) consisting of 0.5% defatted soy bean, 2% malt extract, and 1% KH2PO4. Unless otherwise specified, E. coli cultures were grown in Luria –Bertani (LB) medium supplemented with 0.2% (w/v) maltose and 10 mM MgSO4 at 37 jC. As required, LB medium was supplemented with 50 Ag/ml of kanamycin or 100 Ag/ml of ampicillin. 2.2. Purification of A. sojae LAP and amino acid sequencing The commercially available A. sojae LAP (Corolase LAP, ro¨hm enzyme, Darmstadt, Germany) was filtered through Millipore 25 mm Millex-HA filter (pore size, 0.22 Am; Millipore, Bedford, MA). The resulting material was loaded onto a Hi-Load 26/10 Q Sepharose High Performance column (Amersham Pharmacia Biotech, Buckinghamshire, England) and chromatographed by a Pharmacia AKTA Purifier 10 system. The adsorbed LAP was eluted

Aminopeptidase activity was assayed according to the procedure described by Tan and Konings [27] with some modifications. The standard reaction mixture contained 50 mM glycine– NaOH (pH 8.5), 1 mM L-leucine-p-nitroanilide, and an appropriate amount of the enzyme. After 10 min incubation at 50 jC, the reaction was stopped by the addition of acetic acid to a final concentration of 30% and the absorbance at 504 nm was measured. One unit of enzyme activity was defined as the amount of enzyme that hydrolyzes 1 Amol of leucine-p-nitroanilide per minute with p-nitroanilide as the standard. 2.4. Electrophoresis, activity staining, and protein determination Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the buffer system of Laemmli [28] at a constant voltage (100 V) with gels containing 10% polyacrylamide in the separating gel and 4.5% polyacrylamide in the stacking gel. Prior to electrophoresis, the samples were heated at 100 jC for 5 min in dissociating buffer containing 2% SDS and 5% 2-mercaptoethanol. The molecular mass standards used were myosin (200 kDa), h-galactosidase (116.3 kDa), bovine serum albumin (66.2 kDa), glutamate dehydrogenase (55.4 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa). Detection of protein that displayed LAP activity on gel was performed as described by Manchenko [29]. After SDSPAGE, the gel was washed twice with 2.5% (v/v) Triton X100 in 100 mM sodium phosphate buffer (pH 5.8) for 30 min and then immersed in distilled water for 2 min. The gel

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was incubated in staining solution [100 mM sodium phosphate buffer (pH 5.8), 1 mM CoCl2, 0.04% L-leucyl-2naphthylamide, and 0.06% Fast Black K] in the dark at 37 jC until dark blue bands appear. The stained gel was washed with water and then fixed in 7% acetic acid. The quantitative estimation of protein was carried out by the Bio-Rad protein assay kit according to the supplier’s manual with bovine serum albumin as the standard. 2.5. DNA manipulations DNA electrophoresis, RNA electrophoresis, blotting, hybridization, and general recombinant DNA techniques were carried out by standard procedures [30]. Plasmid DNA was isolated from E. coli strains using a Viogene plasmid miniprep kit (Taipei, Taiwan) according to the manufacturer’s protocol. Genomic DNA of A. sojae was prepared as described by Raeder and Broda [31]. The oligonucleotides used were synthesized under the instructions of Oligo analysis software (National Biosciences, MI) with an Applied Biosystems DNA synthesizer model 380 A. DNA sequencing was performed by the dideoxy chain termination method using a Sequenase kit version 2 (U.S. Biochemicals, Cleveland, OH). Analysis of sequence data and sequence comparisons were performed with programs Blast-X [32] from the National Center for Biotechnology Information and PileUp from the GCG package (Genetics Computer Group, Madison, WI).

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to construct a genomic library. To isolate mRNA from A. sojae, spores (1.5  105) were inoculated into 50 ml of complete medium with shaking at 30 jC for 2 days. After incubation, A. sojae mycelia were disrupted with liquid nitrogen and the total RNAs were prepared by the method of Chomczynski and Sacchi [33]. Poly (A) + RNAs were isolated from the total RNAs using the Dynabeads mRNA Purification kit (Dynal A.S., Oslo, Norway). The doublestrand cDNA was subsequently synthesized by ZAP-cDNA synthesis kit (Stratagene) using 6 Ag of total RNA as the template. The synthesized cDNAs were blunted with Pfu DNA polymerase, and ligated with an EcoRI adapter by T4 DNA ligase. The resulting fragments were digested with EcoRI and XhoI, and inserted into the corresponding sites of Uni-ZAP XR vector. The recombinant phagemids were in vitro packaged by the Gigapack III Gold Packaging Extract (Stratagene) to construct a cDNA library. Plaque hybridization was performed as recommended in the supplier’s protocol using a radiolabelled 700-bp probe. Phagemids were excised from positive phage clones as recommended by the manufacturer. 2.8. Sequence accession number The nucleotide sequence of the lap gene from A. sojae and the encoded amino acid sequence have been deposited in the Genebank nucleotide database under accession number AF419160.

2.6. Preparation of a DNA probe by RT-PCR 3. Results and discussion Two oligonucleotides, 5V-GA(C/T)(A/T)(G/C)CGTGCA (A/G)CA(C/T)AA-(C/T)GA(A/G)AC-3Vand 5V-GT(C/ T)TG(A/G)TACGC(A/G)TC(A/T/G/C)A-C(A/T/G)AT(A/ T/G/C)ATC-3Vwere designed and synthesized as the sense and antisense primers according to the N-terminal amino acid sequence of lysylendopeptidase-cleaved fragments, PDSVQHNE, and KVIVDAYCT, respectively. The chromosomal DNA of A. sojae was used a template and PCR amplifications were performed with a Stratagene Robocycler gradient 96 (Stratagene) using the following program: one cycle of 95 jC for 5 min, 30 cycles of 95 jC for 5 min, 50 jC for 1.5 min, and 72 jC for 2 min, and a final extension at 72 jC for 10 min. DNA labeling of the amplified fragments with a Radiprime II random prime labeling kit (Amersham) and a-32P dCTP was performed according to the manufacturer’s instruction.

3.1. Purification and characterization of A. sojae LAP A commercially available A. sojae LAP with a specific activity of 63.7 U/mg was purified to protein homogeneity by a two-step chromatography procedure described in

2.7. Construction and screening of genomic DNA and cDNA libraries The chromosomal DNA from A. sojae was partially digested with Sau3AI, and the 2 – 7 kb DNA fragments were recovered from the agarose gels with Geneclean kit III (Bio101 Inc., La Jolla, CA). The recovered fragments were ligated to BamHI site of EZAP Express vector (Stratagene)

Fig. 1. SDS-PAGE (A) and activity staining (B) analyses of A. sojae LAP. Lanes: M, protein size marker; 1, commercial LAP; 2, the purified LAP.

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H.-C.R. Chien et al. / Biochimica et Biophysica Acta 1576 (2002) 119–126 Table 2 Substrate specificity of the purified LAP Substratea

Relative activity (%)b

Leu-p-nitroanilide Phe-p-nitroanilide Lys-p-nitroanilide Arg-p-nitroanilide Ala-p-nitroanilide Val-p-nitroanilide Pro-p-nitroanilide Glu-p-nitroanilide

100.0 98.6 41.3 20.3 4.4 2.1 0.5 0.5

a

Fig. 2. Effects of pH and temperature on the activities of A. sojae LAP. The effect of pH on LAP activity was performed at 50 jC for 30 min in the mixture containing purified LAP (4.5 U) in 50 mM citrate-phosphate buffer, pH 3 – 7 (-.-), 50 mM potassium phosphate buffer, pH 6 – 8 (-  -), 50 mM Tris – HCl buffer, pH 6.5 – 9 (-E-), and 50 mM glycine – NaOH buffer, pH 8.5 – 10.5 (-n-). The temperature dependence was determined in 50 mM glycine – NaOH buffer (pH 8.5) at different temperature for 30 min.

Materials and methods. The final purification resulted in a yield of 20% and a three-fold increase in specific activity (190 U/mg). SDS-PAGE analysis (Fig. 1A, lane 2) and activity staining (Fig. 1B, lane 2) showed that the purified enzyme migrated as a single band with an apparent molecular mass of approximately 37 kDa. The apparent temperature dependence in a 30 min assay at pH 8.5 is shown in Fig. 2. A. sojae LAP was most active at 70 jC and greater than 80% of maximal activity was seen from 50 to 80 jC. The enzyme was stable ( >90% of the enzyme activity) at temperatures below 30 jC for 3 days. In contrast, only 50% activity was observed after 1-h incubation at 50 jC. As shown in Fig. 2, the enzyme had a broad pH optimum in the range from 7.5 to 10.

All substrates were used at a final concentration of 1 mM. The rate of hydrolysis is expressed as a percentage of the activity compared to that obtained by using Leu-p-nitroanilide as the substrate at 50 jC, where 100% activity corresponds to 4.5 U in reaction mixture. b

The purified enzyme was strongly inhibited by metalchelating agents, such as EDTA and dipicolinic acid. Activity could be restored by the addition of Zn2 + ions, to a lesser extent, Co2 + ion, whereas the Mg2 + ion was ineffective (Table 1). These results collectively suggest that A. sojae LAP is a metalloprotease and Mg2 + ion is not an endogenous cofactor. EDTA-treated enzyme was effectively reactivated upon the addition of 1 mM ZnCl2. A similar feature has been reported for the aminopeptidases from Streptomyces rimosus [34], Streptococcus thermophilus A [35] and Lactobacillus helveticus CNRZ32 [36]. By means of X-ray crystallography, the aminopeptidases from A. proteolytica [19] and S. griseus [20] have been demonstrated to contain a dinuclear metal active site. The observations of Lin et al. [37] suggest that Co2 + ion with rich spectroscopic properties can be used as an ideal substitute for the native Zn2 + ;

Table 1 Effects of various chemical compounds on the hydrolysis of Leu-pnitroanilide by the purified LAPa Compound

Concentration (mM)

Relative activity (%)

None EDTA EDTA + MgCl2 EDTA + ZnCl2 EDTA + CoCl2 Dipicolinic acid Dipicolinic acid + MgCl2 Dipicolinic acid + ZnCl2 Dipicolinic acid + CoCl2

10 10 + 1 10 + 1 10 + 1 5 5+1 5+1 5+1

100 24 5 125 87 3 12 44 31

a The purified enzyme (4.5 U) was incubated with the chloride salt of the metal ions or chelating agents at 30 jC in 50 mM glycine – NaOH buffer (pH 8.5) for 20 min. The metals used were also analyzed by their ability to restore the LAP activity inhibited by chelating agents. The enzyme was incubated at 30 jC in glycine – NaOH buffer (pH 8.5) with 10 mM EDTA or 5 mM dipicolinic acid for 20 min. After the removal of the chelating agent by Centricon YM-10 (Millipore), the divalent metal salt solution was added, and incubation was continued for an additional 20 min before the enzyme assay. Aminopeptidase activity assayed in the absence of metal ions and inhibitors was taken as 100%.

Fig. 3. Southern blot analysis of genomic DNA isolated from A. sojae using the 700-bp PCR product of the lap gene as a probe. Genomic DNA was digested with the restriction enzymes Bgl1 (lane 1), Kpn1 (lane 2), XhoI (lane 3), SphI (lane 4), EcoRI (lane 5), ClaI (lane 6), and BamHI (lane 7).

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however, in our case, a lesser extent in the reactivation of EDTA-treated LAP was observed by the addition of 1 mM CoCl2.

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Substrate specificity was tested using a variety of amino acid-p-nitroanilide derivatives and small peptides (Table 2). It appears that Leu-p-nitroanilide and Phe-p-nitroanilide

Fig. 4. Alignment of the predicted amino acid sequence with some microbial LAPs. Symbols denote: Aspsojap, A. sojae; Agabisap, A. bisporus; Aerpunap, A. punctata; Vibchoap, V. cholerae; Vibproap, V. proteolyticus. The numbers are positions with respect to the N-terminal amino acid of each enzyme. Identical amino acids are shown on a gray background. Asterisks indicate putative Zn ligands. Residues involved in forming the hydrophobic specificity pocket are indicated by solid triangles. Potential N-glycosylation sites are indicated by solid circles. N-terminal amino acid sequences of the mature enzyme and LAP peptides are underlined. Putative cleavage sites determined using the rules of von Heijne [39] and the site between extra sequence and mature protein are indicated by vertical arrows.

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were most efficiently hydrolyzed by LAP. Lys-p-nitroanilide and Arg-p-nitroanilide were also good targets, while other amino acid-p-nitroanilide derivatives were practically resistant to the action of the enzyme. 3.2. Isolation of a LAP-encoding gene from A. sojae The N-terminal amino acid sequences of seven lysylendopeptidase-cleaved fragments were determined. Based on the determined sequences, two oligonucleotides were synthesized and used for the amplification of the corresponding cDNA fragment. The resulting fragment was 700 bp in size and its sequence was homologous with Agaricus bisporus and Vibrio proteolyticus LAPs. To clone the genomic DNA encoding A. sojae LAP, the 32P-labelled PCR fragment was used as the hybridization probe. Among f 5  104 plaques of the Ec1857 Sam7 genomic library, three positive clones were identified by plaque hybridization. Southern hybridization of genomic DNA digested with BamHI, EcoRI, and PstI using the 0.7-kb fragment as a probe gave one band, suggesting that there is a single copy of the lap gene in the genome of A. sojae (Fig. 3). The 0.7-kb fragment was also used to probe the A. sojae cDNA library. Four colonies produced strong hybridization signals with the probe. A full-length sequence was obtained from one of the clones. An alignment and analysis between the lap gene and its corresponding cDNA revealed the presence of three introns of 59, 68, and 56 bp located at 154– 212, 640– 707, and 1025 – 1080 bp, respectively, downstream of the start codon, ATG. The splice sites of these introns match the fungal consensus sequence, GT –AT boundaries and internal consensus for lariat formation [38]. The lap gene has an open reading frame encoding 377 amino acid residues with a calculated molecular mass of 40.061 kDa. The N-terminal region of the predicted polypeptide revealed a potential signal sequence of 18 amino acids, ending in Ala-Leu-Ala. The alanine residues found at positions 3 and 1 (relative to the putative signal sequence cleavage site) were consistent with the ‘‘( 3, 1)’’ rule of signal sequence [39]. The presence of a putative signal peptide suggests that the LAP is an extracellular protein. We also found additional 59 amino acids (corresponding to amino acid 19 to amino acid 77) in the N-terminal end not found for the purified LAP. Similar incidence also takes place for a phospholipid transfer protein from A. oryzae [40] in which it has a signal peptide of 21 amino acids followed by an extra sequence of 16 amino acids. The functional role of the extra N-terminal sequence in A. sojae LAP, however, remains to be elucidated. The sequences of four lysylendopeptidase-cleaved fragments were found to correspond to 78– 90, 101– 118, 127 –140, and 288 – 307, respectively, of the deduced amino acid sequence (Fig. 4). Based on the determined N-terminal sequence of the purified LAP (corresponding to amino acids 78 –90; Fig. 4), amino acid 78 is the first amino acid of the purified LAP (Fig. 4). The purified LAP is predicted to be a polypeptide of 300 amino acid residues with a molecular mass of 36.28 kDa,

which is similar to that seen in SDS polyacrylamide gel electrophoresis of the deglycosylated enzyme (36 kDa). The 5V-noncoding region contains TATA- and CAAT-like elements at 120 and 286 nucleotides, respectively, upstream of the start codon. The preference for A at position 3, which has been reported in many genes of filamentous fungi [41], was also observed in the lap gene. The putative polyadenylation signals (ATAAAA) are found at 198– 203 bp downstream of the stop codon, UGA. Although the recognition sequences of major nitrogen regulatory protein, NRE [42], and those of carbon catabolite repressor, CREA [43], were not found in the upstream sequence of lap gene, two potential PACC binding consensus sites (5V-GCCARG3V) [44,45] were present at 125 and 135 nucleotides, respectively, within the first 302 bp upstream from the start codon. PACC binding sites have been found in the promoters of the alkaline-expressed ipn A, acv A, and pacC genes, and are involved in the pH regulation of gene expression [45,46]. The presence of PACC binding sites in the A. sojae lap promoter suggests that lap gene is an alkaline-expressed gene and ambient pH may play an important role in the LAP production in A. sojae. 3.3. Amino acid sequence comparison A protein database search revealed that amino acid sequence of the A. sojae LAP has 33.1%, 25.9%, 23.4%, and 23.7% identities with LAPs from A. bisporus (Genebank

Fig. 5. Phylogenetic relationship of the A. sojae LAP with other LAPs based on the sequence alignment. Construction of an unrooted phylogenetic tree is inferred from the alignment of the amino acid sequences by using the ClustalW program (http://www.clustalw.genome.ad.jp/). GenBank accession numbers for the sequences shown here are A. punctata (AB015725), A. bisporus (AJ271690), Aquifex aeolicus (AE000772), Arabidopsis thaliana (X63444), Cattle kidney (S65367), Chlamydia muridarum (AE002299), Chlamydophila pneumoniae (AE002199), Leishmania major (AL117384), Lycopersicon esculentum (U50152), Mesorhizobium loti (AP003012), Mycoplasma pulmonis (AL445564), Petroselinum crispum (X99825), P. aeruginosa (AE004800), Pseudomonas putida (AJ010261), Solanum tuberosum (X77015), Synechocystis sp. (D90911), V. cholerae (D84215), and V. proteolyticus (Z11993).

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AJ271690), Aeromonas punctata (Genebank BAA31158), Vibrio cholerae [47], and V. proteolyticus (previously classified as A. proteolytica) [48], respectively (Fig. 4). Phylogenetic analysis (Fig. 5) indicated that there is only 11 – 33.1% amino acid identity with those from 19 organisms. Of these LAPs, A. bisporus LAP has the highest homology. Perhaps the Agaricus and Aspergillus enzymes are more similar than others because they are both fungi, not because lateral gene transfer occurred. A high conservation is seen in the central region between A. sojae LAP (corresponding to 165 –262 amino acid residues) and aminopeptidases of other species: P. aeruginosa (33%), Mycobac-terium tuberculosis (34%), E. coli K12 (24%), Anabaena variabilis (26%), and S. griseus (31%). A low identity (14%) is found between the A. sojae LAP and a nonspecific aminopeptidase from A. oryzae [49]. The crystal structure of A. proteolytica LAP shows that the active site consists of a metal binding site and a welldefined hydrophobic specificity pocket [19,50]. In addition, the structure of S. griseus apo-LAP exhibits very little change in the geometry of the active site and of the overall structure [20,51]. Such observations suggest that zinc cations do not play a significant structural role in the S. griseus enzyme; rather, they may function in substrate binding and/ or chemical catalysis. The amino acid residues that form the metal binding sites (His-176, Asp-194, Glu-233, Asp-261, and His-343) and a hydrophobic substrate-binding pocket (Cys-310, Tyr-312, Cys-314, Phe-335, and Ile-342) in the A. proteolytica LAP [19] are conserved in other enzymes, including that from A. sojae (Fig. 4). This suggests that all are metalloproteases. 3.4. Deglycosylation After treatment with peptide-N-glycosidase that could remove the N-linked glycans, the purified LAP was analyzed by SDS-PAGE. As shown in Fig. 6, a band with about 36 kDa was seen, indicating that about 1 kDa was removed by peptide-N-glycosidase. A monosaccharide analysis of the released N-linked glycans by HPAEC suggested the presence of oligomannose-type oligosaccharide composed of only mannose and N-acetylglucosamine (data not shown). Posttranslational modification of proteins by glycosylation is an important process that affects their conformation, stability, secretion, and biological activity. N-glycans occur on asparagine residues of mature proteins in almost all eukaryotic cells. It has been speculated that the glycosylation occurs in X-Asn-X-Thr motif of glycoproteins [52]. In this study, two possible sites for N-linked glycosylation are identified in Asn-87 and Asn-288 of A. sojae LAP. In conclusion, we have purified a LAP from A. sojae with a substrate preference on hydrophobic amino acid-pnitroanilides and basic amino acid derivatives. By use of oligonucleotide probe based on the internal amino acid sequences of the purified enzyme, the genomic DNA with three introns and cDNA were cloned. The lap gene consists

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Fig. 6. Electrophoretic analysis of purified A. sojae LAP with and without digestion with peptide-N-glycosidase F. LAP (50 Ag) was denatured under reducing conditions for 3 min at 100 jC and then incubated in the presence (lane 2) or absence (lane 1) of 0.7 units of peptide-N-glycosidase F at 37 jC for 20 h. The protein was precipitated with 4 volumes of ice-cold acetone and then analyzed by 10% SDS-PAGE. Lane M: protein size marker.

of a 18-amino-acid signal peptide, an extra-sequence of 59 amino acids and a 300-amino-acid mature protein with two N-glycosylation sites, Asn-87 and Asn-288. The regulation and physiological role of A. sojae lap gene is now under study in our laboratory. To our knowledge, this is the first report on the characterization and molecular cloning of a LAP from the genus Aspergillus and will benefit to future industrial processes in LAP production.

Acknowledgements This work was supported by grants 89-2.1-64(6) and 902.1.1-Z4(3) from the Council of Agriculture of the Republic of China.

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